In the last reporting period we continued our experiments characterizing the role of miRNA396 in cyst nematode infection. This microRNA targets a group of important transcription factors (GRFs) and we have identified which of these transcription factors are involved in the cyst nematode infection of Arabidopsis. A manuscript detailing these findings has been published. We are working aggressively to transfer this knowledge to the soybean-SCN system. For this purpose, we have developed various strategies to identify and characterize miRNA396 gene family as well as their potential targets in soybean. In soybean (Glycine max), we have established the gene families for the G. max microRNA396 (gma-miR396) and the Growth-Regulating Transcription Factors (GmGRFs). The gma-miR396 gene family consists of eleven functional isomiRs, while the GmGRF family consists of twenty-five. Out of these eleven, we have confirmed expression of seven of the gma-miR396 isomiRs in soybean roots, including both precursor and mature miRNAs, as well as the majority of GmGRFs. In soybean cyst nematode (SCN)-infected soybean roots, we found that all of the GmGRFs that were found to be expressed in roots are significantly up-regulated at a time-point corresponding to feeding site (syncytium) formation. Furthermore, a select suite of GmGRFs were found to be up-regulated significantly higher than the rest of the gene family at this time-point. These GmGRFs were then selected and included in a complete time-course gene expression analysis with both the precursor and mature miR396 isomiRs. We found that at the time-point corresponding to syncytium formation, almost all gma-miR396 isomiRs are significantly down-regulated, while the GmGRFs are significantly up-regulated. Furthermore, at time-points corresponding to syncytium maintenance (i.e. after the formation phase), almost all gma-miR396 isomiRs are significantly up-regulated, while almost all of the selected GmGRFs are significantly down-regulated. We then tested to see if the small suite of GmGRFs is post-transcriptionally regulated by gma-miR396 at the time-point where GmGRFs are significantly down-regulated and gma-miR396 significantly up-regulated (i.e. during syncytium maintenance). As a result, two of the GmGRFs were found to be post-transcriptionally regulated by gma-miR396 at this time-point, one of which is among the most highly expressed GmGRFs in soybean roots. We have also determined that at least some of the gma-miR396 isomiR promoters are activated in the syncytium during the maintenance phase, and thus it is likely that the results obtained from our Q-PCR analyses represent regulation that occurs in syncytia. Finally, we have preliminary evidence in soybean hairy roots that shows that placing the gma-miR396 isomiRs under the control of an SCN-inducible promoter (GmNHL1), which we have shown is active specifically in syncytia of soybean hairy roots during the entire sedentary life cycle, results in significantly decreased susceptibility to SCN. For the coming quarter we are focusing on following strategy to advance our research further.
To conclude whether the Q-PCR results detailed above represent syncytium-specific regulation, we are currently conducting time-course promoter analyses on a select few gma-miR396 isomiRs as well as the GmGRF that was found to be among the most highly expressed in roots, among the most significantly altered during SCN-infection, and found to be post-transcriptionally regulated by gma-miR396 in roots during syncytium maintenance. In order to determine the maximum SCN phenotype that may result from manipulation of the gma-miR396-GmGRF regulatory module, we are constitutively expressing (i.e. under the control of the GmUBI promoter) gma-miR396 isomiRs as well as a miR396-target site mutant of the above mentioned GmGRF in soybean hairy roots, and will evaluate for these potential changes in susceptibility relatively soon. We are also working to obtain conclusive results for manipulating the gma-miR396 isomiRs by placing them under the control of the SCN-inducible promoter – the severity of the phenotypes from our preliminary data may be stronger than we previously observed. It is important that the latter analyses be conclusive for the following purpose – we have received stable transgenic soybean lines that harbor the GmNHL1-miR396 expression constructs for all seven gma-miR396 isomiRs. These transgenic soybean lines may be, one, substantially less susceptible to SCN, whether infected with a virulent or avirulent line, and developmentally normal since we are manipulating the gma-miR396-GmGRF regulatory module specifically in synctyia during SCN infection. Soon we will be characterizing them for nematode phenotype.
In the RNA silencing project targeting fungi, we have completed sequencing of the genes that are expressed in the haustorium of Soybean Rust (SBR) and we have used a series of criteria to computationally identify a set of secreted proteins that are expressed in haustoria the findings of which have been recently published in a peer reviewed research journal (publication is listed below). Currently we are following multiple approaches to advance our knowledge about these potential effectors and to test whether targeting them by using RNA silencing can be used as novel control measures. We have previously cloned and confirmed the sequence of 82 SBR candidate effectors. We have devised a strategy to insert a ~300 base pair fragment of each of these 82 candidate effectors into a Bean pod mottle virus vector (BPMV) and have started making the constructs. These BPMV constructs will be used to produce the silencing signal in soybean plants, which will subsequently be inoculated with SBR. We are also continuing to focus on one effector, PpEC23, as it has suppressed plant immune responses consistently in various assays. In non-host Nicotina benthamiana, we have confirmed that PpEC23 protein is able to suppress the pathogen-inductive expression of several plant immune marker genes using real-time PCR. The fact that it strongly interacts with a soybean protein GmSPL12L, which belongs to a large protein family of transcriptional factors involved in many developmental processes, makes it very interesting candidate to characterize in detail. We have confirmed this interaction both in vitro and in vivo using multiple techniques including yeast-2 hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation. We have utilized BPMV-VIGS technology to transiently silence GmSPL12L in soybean. The GmSPL12L-silencing in soybean resulted into smaller leaves and shorter stature compared to the controls, suggesting its potential involvement in developmental process. When these silenced plants were challenged with Downy Mildew fungus, fewer lesions were observed, indicating GmSPL12L is probably a suppressor of plant immunity. Currently we are testing if the silencing of PpEC23 and/or GmSPL12L alters plant susceptibility to soybean rust.
Publication: Link, T. I., Lang, P., Scheffler, B. E., Duke, M. V., Graham, M. A., Cooper, B., Tucker, M. L., van de Mortel, M., Voegele, R. T., Mendgen, K., Baum, T. J., and Whitham, S. A. (2014) The haustorial transcriptomes of Uromyces appendiculatus and Phakopsora pachyrhizi and their candidate effector families. Mol. Plant Pathol. 15:379-393. doi: 10.1111/mpp.12099